Herpes simplex virus causes human diseases, ranging from cold sores to more serious infections, some of which can be life threatening. Our long term goal has been to delineate the mechanisms that govern HSV entry and HSV induced cell fusion. In contrast to most viruses, HSV utilizes multiple proteins in a multi-step process for fusion and entry. Characterization of these events will likely identify novel paradigms for virus-mediated fusion in general. Four HSV glycoproteins, gD, gB, gH and gL are essential for entry. gD is the receptor binding protein for HSV, while gB and the gH/gL complex form the core fusion machinery for all herpesviruses. Crystal structures of gB, gD and gD bound to its receptors have provided critical insights about how these proteins function in virus entry and cell-fusion. The structural homology of gB with G, the fusion protein of VSV, suggests that gB is a fusion protein in all herpesviruses. Unlike G however, gB does not function on its own but requires gH/gL. Likewise, prior studies showed that gH/gL is a poor fusogen on its own. The prevailing model, based largely on sequence motifs, synthetic peptides and mutations, is that the combination of these two poor fusogens accomplish fusion by acting together in the same membrane (as in virions) to promote fusion. In collaboration with Dr. E. Heldwein, we recently solved the crystal structure of gH/gL. Surprisingly, gH/gL has a novel architecture distinct from any known viral fusion protein, strongly arguing against its role as a fusogen. This structure implies that gB is the only entry protein of HSV that is intrinsically capable of causing fusion. We therefore suggest that gH/gL has a very different role. We postulate that gH/gL functions as a regulator of fusion by binding to gB and activating it into a fusogenic state. In support of this hypothesis, we discovered that the gH/gL ectodomain itself (not membrane bound) can trigger fusion of receptor-bearing cells transfected with gD and gB. In Aim 1, we will dissect the nature of the complex formed by gH/gL with gB with mutants and monoclonal antibodies. Techniques such as biosensor analysis, calorimetry, cryo-EM tomography and X-ray crystallography will be used to gain information about the stoichiometry and conformation of the complex. Bimolecular fluorescence complementation (BiMC) and fusion assays will be used to analyze complex formation when gB and gH/gL are in the same cell (cis) and in different cells (trans). We will determine the ability of mutant forms of gH/gL to trigger gB into a fusogenic state. In Aim 2, we will test gH/gL as a regulator of fusion by varying such parameters as rate and concentration of the WT and mutant forms of soluble gH/gL. We will use BiMC to follow and compare gB-gH/gL complex formation during HSV entry when it occurs by direct fusion at the plasma membrane or by endocytosis. Our work on HSV entry and fusion has strong clinical significance, as each step is a potential target for therapeutics and vaccines against HSV-mediated disease. To test our main hypothesis, we propose two specific aims: 1) to further characterize gH/gL and dissect the nature of the complex it forms with gB; 2) To determine how gH/gL activates gB into a fusogenic state.